Project Summary/Abstract: Chronic obstructive pulmonary disease (COPD), the fourth leading cause of death in the world, is a chronic inflammatory disease thought to be driven at least in part by derangements of protease-antiprotease balance. Recent discoveries have suggested that proteases such as neutrophil elastase (NE) can be associated with exosomes released by neutrophils (PMNs). Exosomes are nanovesicles released by the cell into the environment. NE is enzymatically active when associated with exosomes yet it is markedly resistant to inhibition by ?-1 antitrypsin (?-1AT) in this form. Exosomal NE is several log-fold more potent in causing COPD in mouse models than soluble NE, and the exosomes themselves are able to traverse tissue planes and bind to structural proteins in the lung such as type I collagen, focusing the proteolysis upon its substrate. Importantly, exosomes derived from PMNs that are capable of causing NE-dependent alveolar destruction in mice have been found in bronchoalveolar lavage fluid (BALF) of subjects with COPD, but not healthy controls. Because of these findings, it seems likely that this exosome-associated form of NE may be more important to extracellular matrix destruction (and thus COPD pathogenesis) than conventionally measured soluble NE. This research project will quantify the PMN-derived, NE+ exosome burden within three well-phenotyped cohorts of subjects with COPD as well as non-obstructed never smoker and current/former smoker control subjects. Furthermore, the PMN-derived, NE+ exosome profile of sputum and serum will be assessed to define the biomarker utility of this process using less invasively derived specimens than BALF. These findings will be correlated with various clinically validated measures of COPD disease progression, symptom burden as well as disease sub-phenotype and will be correlated with development of COPD among subjects at risk for this disease. Moreover, this project will develop and optimize an animal model of pathogenic exosome transfer, delineate the mechanism of NE binding to the PMN exosome surface, and advance rational strategies designed to interrupt exosome pathogenicity. These studies will form the foundation for the use and further study of PMN-derived, NE+ exosomes as biomarkers of disease activity and potential therapeutic targets.